The generation of force to produce muscle shortening takes place during the hydrolysis of ATP in a muscle fiber. The mechanism of converting the chemical energy liberated from ATP hydrolysis into mechanical work (energy transduction) involves structural changes in the myosin cross-bridge. The nature of these changes and how they make muscle work is the central question in muscle research. The purpose of the proposed experiments is to elucidate the mechanism of the actomyosin interaction and how it pertains to muscle shortening by determining the relative movements of myosin and actin during contraction. We investigate rigid body or intramolecular movements of myosin by using direction reporting extrinsic probes that chemically modify selected side chains on the cross-bridge, and, fluorescence polarization (FP) and/or electron paramagnetic resonance (EPR) spectroscopy to detect probe orientation. The sensitivity of probes (optical or EPR) to protein rotation depends critically on the orientation of the probe in the protein-fixed reference frame due to ambiguity in the orientation detection techniques. We may reduce the ambiguity by combining data from different probes that are variously oriented relative to the protein using a proven formalism for mapping global rotations of proteins in assemblies. We will extend this method by: (i) investigating local order changes by correlating data from probes of spatially separated sites, (ii) boosting the angular resolution of the probe angular distributions to the theoretical limit, and (iii) introducing and studying the structure of a new class of probes that are luminescent and paramagnetic (L/P). To elucidate the mechanism of protein interaction that results in muscle shortening we will rely on our increasingly well resolved maps of steady-state cross-bridge order and on our ability to distinguish local order changes from global changes. FP and EPR have time-resolved analogues to investigate rotational dynamics of cross-bridges on the submillisecond time domain. Polarized fluorescence photobleaching recovery (PFPR) induces an oriented subset of photobleached probes into the total set of fluorescent probes by an intense pulse of polarized light. The bleached region is monitored with attenuated polarized light and the fluorescence recovery is observed as unbleached fluorophores rotate and their dipoles align with the polarization of the monitoring light. Time-Resolved EPR (TEPR) uses L/P probes that form free radicals when illuminated with light. An intense pulse of polarized light causes the photoselection of an oriented subset of radicals. The TEPR signal changes as the oriented spins rotate. For PFPR and TEPR the signal intensity relaxes to its steady-state value in a time characteristic of probe mobility. PFPR and TEPR investigate probe movement in the three degrees of rotational freedom. The combination of static and dynamic probe order information provides a comprehensive description of the actomyosin interaction.